Platforms for the identification of GPCR targets, and of orthosteric and allosteric modulators

Areas covered in this review: The review provides a summary of old and new approaches for GPCR target identification and for the screening of molecules acting on GPCR targets. The new findings in the field are presented as well as an opinion about how these developments may help GPCR drug discovery. Importance in the field: GPCRs have been the most useful family of proteins in terms of targets for drug discovery. The expectations for GPCR target identification and discovery of new drugs acting on 'old' or 'new' GPCR targets are very high. Given the fact that the pace at which new 'GPCR drugs' appear in the market is decreasing and since the new developments in the field are not being translated into drug discovery there is a need to review the field from a critical perspective. Take home message: To overcome the limitation of the old approaches used in GPCR target identification and drugs discovery new approaches are required. In particular successful approaches in GPCR drug discovery should take into account that the real GPCR targets for a given disease are not GPCR monomers but GPCR heteromers. What the reader will gain: The reader will gain an overview of the strategies currently used and their pros and cons. The reader will also understand that new strategies may help in accelerating the access of GPCR into the market, and also notice that successful strategies should take advantage of the new findings in the field of GPCRs.     

The structure and dynamics of GPCR oligomers: a new focus in models of cell-signaling mechanisms and drug design

G protein-coupled receptors (GPCRs) represent approximately half of the potential pharmaceutical targets for current drugs, and thus the way in which these receptors assemble into dimeric/oligomeric structures is of vital interest in practical as well as conceptual aspects of current drug discovery efforts. The significance of such structures is based on the recent realization that ligand-dependent signaling by GPCRs is not necessarily transduced to the G protein by receptor monomers, but possibly by GPCR dimers or even oligomers that function as dynamic macromolecular assemblies. In addition, recent evidence that GPCR hetero-oligomerization can produce signaling units with unexpected combinations of pharmacological properties suggests entirely new methods for developing successful drugs. The dynamic mechanisms of these signaling assemblies remain to be elucidated. The development of increasingly accurate dynamic molecular models of GPCR dimers is expected to produce a more complete structural context for understanding the molecular mechanisms of GPCR function, and to aid in drug discovery.     

Techniques: promiscuous Galpha proteins in basic research and drug discovery

Assay technologies that measure the activation of heterotrimeric (alphabetagamma) G proteins by G-protein-coupled receptors (GPCRs) are well established within the pharmaceutical industry, either for pharmacological characterization or for the identification of natural or surrogate receptor ligands. Despite recent evidence indicating that GPCR-linked signalling events might not be mediated exclusively by G proteins, G-protein activation remains a common benchmark for assessing GPCR family members. Thus, assay systems that translate ligand-mediated modulation of GPCRs into G-protein-dependent intracellular responses still represent key components of both basic research and the drug discovery process. In this article, the current knowledge and recent progress of integrating Galpha subunits into assay systems for GPCR drug discovery will be reviewed. Emphasis is given to novel promiscuous and chimeric Galpha proteins. Because of their ability to interact with a wide range of GPCRs, such novel G proteins are likely to be incorporated rapidly into drug discovery programmes.     

The significance of G protein-coupled receptor crystallography for drug discovery

Crucial as molecular sensors for many vital physiological processes, seven-transmembrane domain G protein-coupled receptors (GPCRs) comprise the largest family of proteins targeted by drug discovery. Together with structures of the prototypical GPCR rhodopsin, solved structures of other liganded GPCRs promise to provide insights into the structural basis of the superfamily's biochemical functions and assist in the development of new therapeutic modalities and drugs. One of the greatest technical and theoretical challenges to elucidating and exploiting structure-function relationships in these systems is the emerging concept of GPCR conformational flexibility and its cause-effect relationship for receptor-receptor and receptor-effector interactions. Such conformational changes can be subtle and triggered by relatively small binding energy effects, leading to full or partial efficacy in the activation or inactivation of the receptor system at large. Pharmacological dogma generally dictates that these changes manifest themselves through kinetic modulation of the receptor's G protein partners. Atomic resolution information derived from increasingly available receptor structures provides an entrée to the understanding of these events and practically applying it to drug design. Supported by structure-activity relationship information arising from empirical screening, a unified structural model of GPCR activation/inactivation promises to both accelerate drug discovery in this field and improve our fundamental understanding of structure-based drug design in general. This review discusses fundamental problems that persist in drug design and GPCR structural determination.     

Characterization of CHO cells stably expressing a G alpha 16/z chimera for high throughput screening of GPCRs

G protein-coupled receptors (GPCRs) are important therapeutic targets for drug discovery. The identification and characterization of new ligands ideally requires the use of high throughput assays that are applicable to all GPCR subtypes. To circumvent the problem of different GPCRs coupling to distinct intracellular second messenger pathways, we describe a new method that uses the chimeric Galpha protein 16z25 to facilitate this process. Stably expressed in Chinese hamster ovary cells, 16z25 allows G(i/o)- and G(s)-coupled receptors to mobilize intracellular Ca(2+) upon agonist stimulation. We have generated nine cell lines each stably expressing 16z25 and a GPCR. All cell lines respond to appropriate agonist stimulation in fluorometric imaging plate reader (FLIPR) assays with robust and potent Ca(2+) mobilization. Several of these lines have been pharmacologically characterized using agonists and antagonists. We also demonstrate that the coexpression of GPCR and 16z25 does not interfere with the receptors' ability to activate endogenous signaling pathways. The ability of 16z25 to functionally mediate the agonist stimulation of a broad spectrum of GPCRs indicates that the use of cell lines stably coexpressing this chimera and GPCRs will simplify the drug screening process and aid in the deorphanization of new receptors.     

Emerging concepts of guanine nucleotide-binding protein-coupled receptor (GPCR) function and implications for high throughput screening

Guanine nucleotide binding protein (G protein) coupled receptors (GPCRs) comprise one of the largest families of proteins in the human genome and are a target for 40% of all approved drugs. GPCRs have unique structural motifs that allow them to interact with a wide and diverse series of extracellular ligands, as well as intracellular proteins, G proteins, receptor activity-modifying proteins, arrestins, and indeed other receptors. This distinctive structure has led to numerous efforts to discover drugs against GPCRs with targeted therapeutic uses. Such "designer" drugs currently include allosteric regulators, inverse agonists, and drugs targeting hetero-oligomeric complexes. Moreover, the large family of orphan GPCRs provides a rich and novel field of targets to discover drugs with unique therapeutic properties. The numerous technologies to discover GPCR drugs have also greatly advanced over the years, facilitating compound screening against known and orphan GPCRs, as well as in the identification of unique designer GPCR drugs. Indeed, high throughput screening (HTS) technologies employing functional cell-based approaches are now widely used. These include measurement of second messenger accumulation such as cyclic AMP, calcium ions, and inositol phosphates, as well as mitogen-activated protein kinase activation, protein-protein interactions, and GPCR oligomerization. This review focuses on how the improved understanding of the molecular pharmacology of GPCRs, coupled with a plethora of novel HTS technologies, is leading to the discovery and development of an entirely new generation of GPCR-based therapeutics.     

Orphan G-protein-coupled receptors: the next generation of drug targets?

The pharmaceutical industry has readily embraced genomics to provide it with new targets for drug discovery. Large scale DNA sequencing has allowed the identification of a plethora of DNA sequences distantly related to known G protein-coupled receptors (GPCRs), a superfamily of receptors that have a proven history of being excellent therapeutic targets. In most cases the extent of sequence homology is insufficient to assign these `orphan'' receptors to a particular receptor subfamily. Consequently, reverse molecular pharmacological and functional genomic strategies are being employed to identify the activating ligands of the cloned receptors. Briefly, the reverse molecular pharmacological methodology includes cloning and expression of orphan GPCRs in mammalian cells and screening these cells for a functional response to cognate or surrogate agonists present in biological extract preparations, peptide libraries, and complex compound collections. The functional genomics approach involves the use of `humanized yeast cells, where the yeast GPCR transduction system is engineered to permit functional expression and coupling of human GPCRs to the endogenous signalling machinery. Both systems provide an excellent platform for identifying novel receptor ligands. Once activating ligands are identified they can be used as pharmacological tools to explore receptor function and relationship to disease.     

G protein-coupled receptor allosterism and complexing

G protein-coupled receptors (GPCRs) represent the largest family of cell-surface receptors. These receptors are natural allosteric proteins because agonist-mediated signaling by GPCRs requires a conformational change in the receptor protein transmitted between two topographically distinct binding sites, one for the agonist and another for the G protein. It is now becoming increasingly recognized, however, that the agonist-bound GPCR can also form ternary complexes with other ligands or "accessory" proteins and display altered binding and/or signaling properties in relation to the binary agonist-receptor complex. Allosteric sites on GPCRs represent novel drug targets because allosteric modulators possess a number of theoretical advantages over classic orthosteric ligands, such as a ceiling level to the allosteric effect and a potential for greater GPCR subtype-selectivity. Because of the noncompetitive nature of allosteric phenomena, the detection and quantification of such effects often relies on a combination of equilibrium binding, nonequilibrium kinetic, and functional signaling assays. This review discusses the development and properties of allosteric receptor models for GPCRs and the detection and quantification of allosteric effects. Moreover, we provide an overview of the current knowledge regarding the location of possible allosteric sites on GPCRs and candidate endogenous allosteric modulators. Finally, we discuss the potential for allosteric effects arising from the formation of GPCR oligomers or GPCRs complexed with accessory cellular proteins. It is proposed that the study of allosteric phenomena will become of progressively greater import to the drug discovery process due to the advent of newer and more sensitive GPCR screening technologies.     

Fragment-based lead discovery on G-protein-coupled receptors

Introduction: G-protein-coupled receptors (GPCRs) form one of the largest groups of potential targets for novel medications. Low druggability of many GPCR targets and inefficient sampling of chemical space in high-throughput screening expertise however often hinder discovery of drug discovery leads for GPCRs. Fragment-based drug discovery is an alternative approach to the conventional strategy and has proven its efficiency on several enzyme targets. Based on developments in biophysical screening techniques, receptor stabilization and in vitro assays, virtual and experimental fragment screening and fragment-based lead discovery recently became applicable for GPCR targets.

Areas covered: This article provides a review of the biophysical as well as biological detection techniques suitable to study GPCRs together with their applications to screen fragment libraries and identify fragment-size ligands of cell surface receptors. The article presents several recent examples including both virtual and experimental protocols for fragment hit discovery and early hit to lead progress.

Expert opinion: With the recent progress in biophysical detection techniques, the advantages of fragment-based drug discovery could be exploited for GPCR targets. Structural information on GPCRs will be more abundantly available for early stages of drug discovery projects, providing information on the binding process and efficiently supporting the progression of fragment hit to lead. In silico approaches in combination with biological assays can be used to address structurally challenging GPCRs and confirm biological relevance of interaction early in the drug discovery project.     

Tools for GPCR drug discovery

G-protein-coupled receptors (GPCRs) mediate many important physiological functions and are considered as one of the most successful therapeutic targets for a broad spectrum of diseases. The design and implementation of high-throughput GPCR assays that allow the cost-effective screening of large compound libraries to identify novel drug candidates are critical in early drug discovery. Early functional GPCR assays depend primarily on the measurement of G-protein-mediated 2nd messenger generation. Taking advantage of the continuously deepening understanding of GPCR signal transduction, many G-protein-independent pathways are utilized to detect the activity of GPCRs, and may provide additional information on functional selectivity of candidate compounds. With the combination of automated imaging systems and label-free detection systems, such assays are now suitable for high-throughput screening (HTS). In this review, we summarize the most widely used GPCR assays and recent advances in HTS technologies for GPCR drug discovery.     

针对G蛋白偶联受体的药物筛选新方法

G蛋白偶联受体（GPCR）为具有7个跨膜螺旋的蛋白质受体，是人体内最大的蛋白质家族，其为极重要的药物靶点。本文针对GPCR的固有激活和变构效应的药物筛选模型开发新进展和高内涵药物筛选新技术进行综述。     

Computational identification and analysis of G protein‐coupled receptor targets

The completion of the human genome sequence has provided a large pool of potential drug targets for disease therapy. G protein–coupled receptors (GPCRs), which are central to signaling networks that regulate basic cellular processes, represent the most important known class of therapeutic targets for multiple disease states. Bioinformatics approaches can be applied to facilitate the identification of novel GPCRs, understanding their physiological and pathological roles, and screening for drug discovery. The present review summarizes current bioinformatics approaches that can be used to identify and analyze GPCR targets. In addition, the limitations of these technologies with the intention of setting reasonable expectations are also discussed together with some potential avenues for GPCR research. Drug Dev. Res. 67:771–780, 2006. © 2007 Wiley‐Liss, Inc.     

Molecular modeling of vasopressin receptor and in silico screening of V1b receptor antagonists

Introduction: G protein-coupled receptors (GPCRs) are integral membrane proteins which contain seven-transmembrane-spanning alpha-helices. GPCR-mediated signaling has been associated with various human diseases, positioning GPCRs as attractive targets in the drug discovery field. Recently, through advances in protein engineering and crystallography, the number of resolved GPCR structures has increased dramatically. This growing availability of GPCR structures has greatly accelerated structure-based drug design (SBDD) and in silico screening for GPCR-targeted drug discovery.

Areas covered: The authors introduce the current status of X-ray crystallography of GPCRs and what has been revealed from the resolved crystal structures. They also review the recent advances in SBDD and in silico screening for GPCR-targeted drug discovery and discuss a docking study, using homology modeling, with the discovery of potent antagonists of the vasopressin 1b receptor.

Expert opinion: Several innovative protein engineering techniques and crystallographic methods have greatly accelerated SBDD, not only for already-resolved GPCRs but also for those structures which remain unclear. These technological advances are expected to enable the determination of GPCR-fragment complexes, making it practical to perform fragment-based drug discovery. This paves the way for a new era of GPCR-targeted drug discovery.     

Strategies for the identification of allosteric modulators of G-protein-coupled receptors

Once considered a pharmacological curiosity, allosteric modulation of seven transmembrane domain G-protein-coupled receptors (GPCRs) has emerged as a potentially powerful means to affect receptor function for therapeutic purposes. Allosteric modulators, which interact with binding sites topologically distinct from the orthosteric ligand binding sites, can potentially provide improved selectivity and safety, along with maintenance of spatial and temporal aspects of GPCR signaling. Accordingly, drug discovery efforts for GPCRs have increasingly focused on the identification of allosteric modulators. This review is devoted to an examination of the strategies, challenges, and opportunities for high-throughput screening for allosteric modulators of GPCRs, with particular focus on the identification of positive allosteric modulators.     

Crosstalk within GPCR heteromers in schizophrenia and Parkinson's disease: physical or just functional?

Crosstalk between G protein-coupled receptors (GPCRs) is one of the key mechanisms used by the cell for integrating multiple signaling pathways. Functional crosstalk at the level of signaling pathways was initially thought to regulate receptor function. Importantly, the existence of GPCR heteromers demonstrates that direct physical interactions between GPCRs could also be behind the crosstalk phenomenon. Neurological disorders such as Parkinson's disease (PD) and schizophrenia have been linked to a dysfunctional communication between certain GPCRs. In this review, we discuss functional and physical crosstalk of the main GPCR families involved in the aforementioned disorders. In addition, we analyze the available structural information on physical crosstalk and highlight some strategies in drug discovery based on these crosstalk mechanisms.     

X-ray structure breakthroughs in the GPCR transmembrane region

G-protein-coupled receptor (GPCR) proteins [Lundstrom KH, Chiu ML, editors. G protein-coupled receptors in drug discovery. CRC Press; 2006] are the single largest drug target, representing 25-50% of marketed drugs [Overington JP, Al-Lazikani B, Hopkins AL. How many drug targets are there? Nat Rev Drug Discov 2006;5(12):993-6; Parrill AL. Crystal structures of a second G protein-coupled receptor: triumphs and implications. ChemMedChem 2008;3:1021-3]. While there are six subclasses of GPCR proteins, the hallmark of all GPCR proteins is the transmembrane-spanning region. The general architecture of this transmembrane (TM) region has been known for some time to contain seven α-helices. From a drug discovery and design perspective, structural information of the GPCRs has been sought as a tool for structure-based drug design. The advances in the past decade of technologies for structure-based design have proven to be useful in a number of areas. Invoking these approaches for GPCR targets has remained challenging. Until recently, the most closely related structures available for GPCR modeling have been those of bovine rhodopsin. While a representative of class A GPCRs, bovine rhodopsin is not a ligand-activated GPCR and is fairly distant in sequence homology to other class A GPCRs. Thus, there is a variable degree of uncertainty in the use of the rhodopsin X-ray structure as a template for homology modeling of other GPCR targets. Recent publications of X-ray structures of class A GPCRs now offer the opportunity to better understand the molecular mechanism of action at the atomic level, to deploy X-ray structures directly for their use in structure-based design, and to provide more promising templates for many other ligand-mediated GPCRs. We summarize herein some of the recent findings in this area and provide an initial perspective of the emerging opportunities, possible limitations, and remaining questions. Other aspects of the recent X-ray structures are described by Weis and Kobilka [Weis WI, Kobilka BK. Structural insights into G-protein-coupled receptor activation. Curr Opin Struct Biol 2008;18:734-40] and Mustafi and Palczewski [Mustafi D, Palczewski K. Topology of class A G protein-coupled receptors: insights gained from crystal structures of rhodopsins, adrenergic and adenosine receptors. Mol Pharmacol 2009;75:1-12].     

Structure-based drug screening for G-protein-coupled receptors

G-protein-coupled receptors (GPCRs) represent a large family of signaling proteins that includes many therapeutic targets; however, progress in identifying new small molecule drugs has been disappointing. The past 4 years have seen remarkable progress in the structural biology of GPCRs, raising the possibility of applying structure-based approaches to GPCR drug discovery efforts. Of the various structure-based approaches that have been applied to soluble protein targets, such as proteases and kinases, in silico docking is among the most ready applicable to GPCRs. Early studies suggest that GPCR binding pockets are well suited to docking, and docking screens have identified potent and novel compounds for these targets. This review will focus on the current state of in silico docking for GPCRs.